Type 1A diabetes (T1D) is characterized by an absolute insulin insufficiency resulting from autoimmune destruction of pancreatic beta cells, and is the most common autoimmune endocrine disease afflicting children in the US. It is responsible for significant societal and health care costs, and is increasing at a rate of approximately 2 - 5% per year. Diabetes autoantibodies are currently the most robust biomarkers of disease, and are central to diagnosis, and for stratification of those genetically at risk. Autoantibodies to insulin (IAA) are typically the first to develop, especially in young children, and their measurement may enable identification of individuals who are most likely to benefit from prophylactic insulin therapy. This makes a reliable assay for IAAs particularly important. However the presently used assay performs poorly in standardization tests, and is clearly in need of improvement. Our ongoing studies of autoantibodies to zinc transporter 8 (ZNT8A) have demonstrated that significant improvements to assay specificity and sensitivity can be achieved by making modifications to the probe used for their detection, Our hypothesis is that we can apply the experience we have gained from optimizing the ZNT8A assay to develop an improved procedure for measuring IAAs, based upon the use of novel luciferase containing probes. Our preliminary data indicate that IAAs can be detected using a fusion protein between human preproinsulin (hPPI) and a modified Gaussia princeps luciferase (GLuc) that has enhanced light emission stability. The single specific aim of the current proposal is to optimize this hPPI- GLuc based IAA assay. Initially this will involve a systematic investigation of key features of the probe, such as inclusion of multiple domains, potential for excision of the C peptide, and sequence of the insulin component (human v porcine). Subsequently we will optimize probe production and basic features of the assay procedure such as length and temperature of the incubation between diabetic sera and the probe, and the method of collection of the resulting immune complexes. We believe that the current proposal will lead to the development of an assay for IAAs that has at least equivalent specificity and sensitivity to the currently used mIAA, with the greater utility of not involving the use of radio-isotopes. The basic probe design can also be adapted to incorporate other protein antigens, and hence our proposal could have significant utility for the diagnosis of a variety of other autoimmune diseases, in addition to T1D. PUBLIC HEALTH RELEVANCE: Type 1A insulin dependent diabetes mellitus is one of the most frequent chronic diseases of children and young adults, and carries a high risk of devastating complications in later life. Autoantibodies are the most robust biomarkers of disease, and our proposal will develop improved procedures for measuring autoantibodies to insulin, which currently show poor reproducibility. Insulin is one of the major molecular targets in type 1A diabetes, especially in young children, and our proposal will facilitate diagnosis, and the identification of individuals who may benefit the most from preventative insulin therapy.